Erythromycin (EM) and clindamycin (CM) susceptibility testing was performed on 222 clinical isolates of group B Streptococcus. A multiplex PCR assay was used to detect the ermB, ermTR, and mefA/E antibiotic resistance genes. These results were compared to the phenotypes as determined by the standard EM/CM double disk diffusion assay.Group B Streptococcus (GBS) is one of the leading causes of neonatal bacterial infection. This type of infection commonly leads to pneumonia, septicemia, or meningitis. Because of the serious nature of neonatal GBS infections, the suggested standard protocol for the obstetrician/gynecologist is that pregnant women should be tested for the presence of GBS at 35 to 37 weeks of gestation (7,15). Once GBS colonization is diagnosed, the typical treatment for these patients is penicillin, to which there is no known resistance. However, there is a significant population of penicillin-allergic patients, a reported 12% of pregnant women (12), for whom the macrolide (erythromycin [EM]) or lincosamide (clindamycin [CM]) class of drugs needs to be administered, in particular, for those patients who are at high risk for anaphylactic shock. Previous reports have cited resistance of GBS to EM and CM of up to 37% and 17%, respectively (7). The resistance is commonly caused by three genes: ermB, ermTR, and mefA/E (1, 9, 10). The ermB and ermTR genes encode 23S rRNA methylases, which alter the binding of the antibiotic target site. The expression of these genes leads to the constitutively expressed and the erythromycin-induced macrolide, lincosamide, and streptogramin B (cMLS and iMLS, respectively) resistance phenotypes (9). The mefA and mefE genes, which are 90% identical, encode 14-and 15-member macrolide efflux pumps and lead to the macrolide only (M) resistance phenotype (1). Because of the presence of ermB, ermTR, mefA/E, and other antibiotic resistance genes on plasmids and/or transposons, these genes can pass from organism to organism, and the monitoring of the antibiotic resistance of GBS should occur regularly (13). We used a multiplex PCR assay to screen for the prevalence of the ermB, ermTR, and mefA/E genes in GBS clinical isolates from 222 patients for whom physicians ordered GBS testing. The samples, representing 20 states in the United States and 60% of which were from Florida, New Jersey, and Texas, were chosen at random. Patient ages ranged from 15 to 82 years, with an average of 31.3 Ϯ 11.8 years. These results were compared to the antibiotic resistance phenotypes as determined by the standard EM/CM double disk diffusion assay (3,11,15)